EP3939990A1 - Neuartiges zwischenprodukt für biologisch aktives polypeptid und verfahren zur herstellung davon - Google Patents

Neuartiges zwischenprodukt für biologisch aktives polypeptid und verfahren zur herstellung davon Download PDF

Info

Publication number
EP3939990A1
EP3939990A1 EP20756094.7A EP20756094A EP3939990A1 EP 3939990 A1 EP3939990 A1 EP 3939990A1 EP 20756094 A EP20756094 A EP 20756094A EP 3939990 A1 EP3939990 A1 EP 3939990A1
Authority
EP
European Patent Office
Prior art keywords
trt
tbu
resin
boc
fmoc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20756094.7A
Other languages
English (en)
French (fr)
Other versions
EP3939990A4 (de
Inventor
Wonkyoung Choi
Nari Kim
Jonghwan Park
Sungjun Park
Namdu Kim
Youngbum Cho
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hanmi Fine Chemicals Co Ltd
Original Assignee
Hanmi Fine Chemicals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hanmi Fine Chemicals Co Ltd filed Critical Hanmi Fine Chemicals Co Ltd
Publication of EP3939990A1 publication Critical patent/EP3939990A1/de
Publication of EP3939990A4 publication Critical patent/EP3939990A4/de
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the present invention relates to a novel intermediate used for physiologically active polypeptides and a method for preparing the same. More specifically, the present invention relates to a method for preparing a physiologically active polypeptide in a safer and more effective manner, a novel polypeptide intermediate used therefor, and a method for preparing the same.
  • Diabetes-related diseases including obesity and type 2 diabetes, are among the typical metabolic diseases developing in modern society, and are recognized as an important threat to health worldwide. Accordingly, economic costs are also increasing rapidly.
  • Glucagon is produced in the pancreas when the blood glucose levels fall due to reasons such as medications, diseases, deficiency in hormones or enzymes, etc. Glucagon sends a signal for glycogen breakdown in the liver to induce the release of glucose and increases blood glucose to a normal level.
  • glucagon In addition to the effect of increasing the blood glucose levels, glucagon suppresses appetite and activates hormone-sensitive lipase of adipocytes to promote lipolysis, thereby showing an anti-obesity effect. Various studies related to glucagon are underway.
  • Korean Laid-Open Publication No. 10-2017-0080521 discloses a triple agonist having activities to all of glucagon, GLP-1, and GIP receptors and uses thereof.
  • Such a peptide may be composed of substitution, addition, deletion, modification, and combinations thereof in at least one or more amino acids in the native glucagon sequences, and more specifically, it discloses an isolated peptide including the amino acid sequence represented by the following General Formula 1:
  • the peptide may be prepared by way of methods known in the art, for example, synthesis via an automatic peptide synthesizer, genetic manipulation technique, or any other methods, depending on the length of the peptide.
  • synthesis via an automatic peptide synthesizer for example, synthesis via an automatic peptide synthesizer, genetic manipulation technique, or any other methods, depending on the length of the peptide.
  • the quality of high purity, a yield suitable for commercialization, and a production process suitable for mass production are required.
  • one aspect of the present invention provides a novel polypeptide intermediate represented by Chemical Formula 1 below:
  • R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl;
  • X is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl,
  • a to D are protecting groups;
  • a to D are each independently selected from the group consisting of triphenylmethyl (Trt), tert -butyl (tBu), t -butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf);
  • R is selected from the group consisting of H, linear or branched C 1-12 alkyl, linear or branched C 1-12 alkyloxycarbonyl, linear or branched C 2-12 alkenyl, C 3-10 cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl, and heteroaryl;
  • X' is a resin; and these substituents may be
  • a physiologically polypeptide and a pharmaceutically acceptable salt thereof including the steps of:
  • novel polypeptide intermediate and the method for preparing the same according to the present invention can provide a novel polypeptide intermediate that can be used for physiologically active polypeptide pharmaceuticals, and are suitable for mass production and have the advantage of being able to produce efficient and high-quality products with reproducibility.
  • FIG. 1 refers to a chromatogram comparing the distribution of each target compound produced after cleavage of the protecting group and the resin in the Example and Comparative Example.
  • the protecting group of the amino acid used herein may be any one which has stability under the conditions of the peptide condensation reaction, can be easily removed, does not affect the peptide chain and the substituents during the removal reaction, and does not cause racemization of any chiral center present in the peptide.
  • suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4-nitrophenyl-sulfonyl)ethoxycarbonyl (NSC), t -butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl, t -amyloxycarbonyl, isobornyloxycarbonyl, ( ⁇ , ⁇ )-dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl, 2-cyano- t -butyloxycarbonyl, etc ., but are not limited thereto, and other suitable protecting groups known in the art for this purpose may also be used within the scope of the present invention.
  • 9-fluorenylmethyloxycarbonyl (Fmoc) or t -butoxycarbonyl (Boc) may be used.
  • a solid-phase peptide synthesis method using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group may be used among the amino acid protecting groups.
  • the resin used in all steps of the reaction of the present invention is a polymer support treated with an appropriate linker, and a polystyrene (PS)-based resin or a polystyrene-polyethylene glycol copolymer (PS-PEG copolymer)-based resin is preferable, but the resin is not limited thereto, and other suitable resins known in the art for this purpose may also be used within the scope of the present invention.
  • PS polystyrene
  • PS-PEG copolymer polystyrene-polyethylene glycol copolymer
  • the resin that can be used in the present invention may include polystyrene (PS)-based resins such as aminomethyl resin, aminoethyl resin, aminobutyl resin, Rink amide aminomethyl resin, Rink amide aminoethyl resin, Rink amide aminobutyl resin, Rink amide MBHA resin, Rink amide resin, 2-chlorotrityl- N -Fmoc-hydroxylamine resin, HMPA-AM resin, HMPB resin, 2-chlorotrityl resin, 4-carboxytrityl resin, Wang resin, PAL resin, 4-(hydroxymethyl)phenoxyacetic acid resin, and Sieber amide resin, and polystyrene-polyethylene glycol copolymer-based resins such as such as TentaGel S resin, TentaGel R resin, TentaGel XV resin, TentaGel MB resin, TentaGel HL resin, TentaGel B resin, Tenta
  • the polar aprotic solvent used in all steps of the reaction of the present invention may include, but is not limited to, for example, dimethylformamide, dimethylacetamide, etc., and other suitable polar aprotic solvents known in the art for this purpose may also be used within the scope of the present invention.
  • the polar aprotic solvent used in all steps of the reaction of the present invention may be preferably selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  • Another aspect of the present invention provides a method for preparing a resin composite compound of Chemical Formula 3.
  • the method for preparing the resin composite compound of the present invention includes the steps of:
  • step (1) the resin is swollen in a polar aprotic solvent.
  • the deprotected resin may be prepared by removing the protecting group using, for example, a piperidine solution in a polar aprotic solvent.
  • the deprotected resin may be washed using a polar solvent.
  • the polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol, and mixtures thereof.
  • the deprotected resin can be activated.
  • the protected amino acid is activated by adding the protected amino acid, 1-hydroxy-1 H -benzotriazole, and 1,3-diisopropylcarbodiimide in a polar aprotic solvent.
  • step (2) a coupling reaction is performed by adding an activated protected amino acid solution to the deprotected resin in a reactor.
  • the coupled resin may be washed using a polar solvent.
  • the polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol, and mixtures thereof.
  • step (3) the above steps (1)-(2) are repeatedly performed until a peptide is formed.
  • step (3) may be repeated 2 to 100 times until a peptide of a desired length is formed, preferably 10 to 50 times, and most preferably 14 to 30 times.
  • a partially deprotected resin is prepared by reacting the synthesized peptide with tetrakispalladium, N -methylaniline, and phenylsilane in a solvent.
  • the solvent used in the reaction of step (4) may be selected from the group consisting of dichloromethane, chloroform, and mixtures thereof.
  • a polypeptide intermediate is prepared by performing a cyclization reaction by adding the synthesized peptide and a coupling reagent in a polar aprotic solvent.
  • the coupling reagent used in the reaction of step (5) may be selected from 1-hydroxy-1 H -benzotriazole/1,3-diisopropylcarbodiimide or HATU (1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5- b ]-pyridinium 3-oxide hexafluorophosphate)/ N , N -diisopropylethylamine, but is not limited thereto, and other suitable coupling reagents known in the art for this purpose may also be used within the scope of the present invention.
  • Still another aspect of the present invention provides a method for preparing a physiologically active polypeptide represented by Chemical Formula 2 below or a pharmaceutically acceptable salt thereof from the method described above.
  • the method for preparing a physiologically active polypeptide of Chemical Formula 2 and a pharmaceutically acceptable salt according to the present invention may include:
  • step (1) an amino acid linking reaction is repeatedly performed until a peptide having a desired amino acid sequence is formed by reacting the resin composite compound of Chemical Formula 3 obtained by way of the above method or another method with an amino acid.
  • the reaction of step (1) may be repeatedly performed 1 to 50 times until a peptide of a desired length is formed, and preferably 1 to 30 times.
  • the physiologically polypeptide of Chemical Formula 2 is in the form of a trifluoroacetic acid salt or an acetic acid salt.
  • the reaction step (1) described above may include a step of deprotecting the peptide-resin composite in a polar aprotic solvent.
  • the polar aprotic solvent may be selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  • step (2) the desired peptide is cleaved from the resin while deprotecting the protected resin using a cleavage cocktail simultaneously.
  • the cleavage cocktail of step (2) may include a solution of trifluoroacetic acid (TFA), at least one scavenger, and dichloromethane.
  • TFA trifluoroacetic acid
  • scavenger at least one scavenger
  • dichloromethane dichloromethane
  • the scavenger of step (2) may be selected from the group consisting of triisopropylsilane (TIPS), triethylsilane (TES), phenol, anisole, thioanisole, water, ethanedithiol (EDT), 1-dodecanethiol, dithiothreitol (DTT), and indole, but is not limited thereto, and other suitable scavengers known in the art for this purpose may also be used within the scope of the present invention.
  • TIPS triisopropylsilane
  • TES triethylsilane
  • phenol anisole
  • thioanisole water
  • EDT ethanedithiol
  • DTT dithiothreitol
  • indole indole
  • the preparation method of the present invention may further include a step of filtering the cleaved mixture from the resin after step (2).
  • the preparation method of the present invention provides the compound of Chemical Formula 1 and the resin composite compound of Chemical Formula 3, which are novel cyclized polypeptide intermediates that can be used as high-purity pharmaceutical intermediates.
  • the cyclized intermediate of the present invention is used, the improvement in yield and inhibition in impurity generation may be simultaneously achieved in the preparation of physiologically active polypeptides.
  • the generated related substances are changed to a type that can be easily separated from the product, so that the final purification can be easily performed to thereby obtain high-quality pharmaceuticals.
  • novel polypeptide intermediate according to the present invention and the physiologically active polypeptide prepared by way of the preparation method thereof are subjected to a cyclization reaction after preparing a linear polypeptide up to 15mer, and the remaining amino acids are further synthesized.
  • the preparation method of the present invention also improves the generation of related substances, and thus, the final purification process after completion of synthesis can be carried out easily, and the components of heavy metals can be easily managed. This is a beneficial effect beyond what is expected when simply changing the position of the lactam cyclization process, assuming that other factors remain unchanged.
  • the preparation method of the present invention has the advantage being an efficient process suitable for commercial production because the overall yield and purity are greatly improved.
  • amino acids referred to by abbreviation herein have been described according to the IUPAC-IUB nomenclature.
  • SPPS methods Solid-phase peptide synthesis methods, including methods for deprotecting amino acids, methods for cleaving peptides from resins, and purification thereof), as well as methods for detection and characterization of the resulting peptides (LCMS, MALDI, and UPLC) method) are related.
  • the protected amino acid derivative used is the standard Fmoc-amino acid.
  • the alpha amino group at the N-terminal amino acid has been protected with Boc (e.g., Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides having His at the N-terminus).
  • Rink amide MBHA resin 80.0 g (0.31 mmol/g) of Rink amide MBHA resin and 480 mL of dimethylformamide were added to a vessel, stirred for 15 minutes, and then filtered to remove dimethylformamide. This process was performed twice. Through the above process, a Rink amide MBHA resin was prepared.
  • Cys(Trt)-Fmoc activated in step (2) was added to the Fmoc-deprotected Rink amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Fmoc, was obtained.
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • Thr(tBu)-Fmoc In a vessel, 29.6 g of Thr(tBu)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Thr(tBu)-Fmoc, was activated.
  • Thr(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Thr(tBu)-Fmoc(C), was obtained.
  • Step (2) Activation of Asn(Trt)-Fmoc(N)
  • Asn(Trt)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc, was obtained.
  • Met-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc (Synthesis of Poly 5mer)
  • Leu-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc, was obtained.
  • Trp(Boc)-Fmoc In a vessel, 39.2 g of Trp(Boc)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Trp(Boc)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc (Synthesis of Poly 6mer)
  • Trp(Boc)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc, was obtained.
  • Step (2) Activation of Gln(Trt)-Fmoc(Q)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc (Synthesis of Poly 7mer)
  • Gln(Trt)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc, was obtained.
  • Step (2) Activation of Val-Fmoc(V)
  • Val-Fmoc In a vessel, 45.4 g of Val-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Val-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc (Synthesis of Poly 8mer)
  • Val-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • Step (2) Activation of Phe-Fmoc(F)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Fmoc (Synthesis of Poly 9mer)
  • Phe-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • Glu(OtBu)-Fmoc In a vessel, 31.6 g of Glu(OtBu)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Glu(OtBu)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Fmoc (Synthesis of Poly 10mer)
  • Glu(OtBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu) was obtained.
  • Step (2) Activation of Lys(Alloc)-Fmoc(K)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc (Synthesis of Poly 11mer)
  • Lys(Alloc)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • Step (2) Activation of Ala-Fmoc(A)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Fmoc (Synthesis of Poly 12mer)
  • Ala-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala, was obtained.
  • Step (2) Activation of Arg(Pbf)-Fmoc(R)
  • Arg(Pbf)-Fmoc In a vessel, 48.3 g of Arg(Pbf)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Arg(Pbf)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Fmoc (Synthesis of Poly 13mer)
  • Arg(Pbf)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf), was obtained.
  • Step (2) Activation of Lys(Boc)-Fmoc(K)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Fmoc (Synthesis of Poly 14mer)
  • Lys(Boc)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc) was obtained.
  • Step (2) Activation of Glu(OAII)-Fmoc(E)
  • Glu(OAII)-Fmoc In a vessel, 30.5 g of Glu(OAII)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Glu(OAII)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc (Synthesis of Poly 15mer)
  • Glu(OAII)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • the cyclization step can be carried out by way of the process of either (2)-1 or (2)-2 below.
  • Step (2)-1 Preparation of Cyclized Polypeptide (Synthesis of Cyclized Poly 15mer)
  • Step (2)-2 Preparation of Cyclized Polypeptide (Synthesis of Cyclized Poly 15mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Fmoc, was obtained.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu], was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Fmoc (Synthesis of Cyclized Poly 16mer)
  • Asp(OtBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu] prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Fmoc (Synthesis of Cyclized Poly 17mer)
  • Leu-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc (Synthesis of Cyclized Poly 18mer)
  • Tyr(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc, was obtained.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu), was obtained.
  • Step (2) Activation of Lys(Boc)-Fmoc(K)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc (Synthesis of Cyclized Poly 19mer)
  • Lys(Boc)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc, was obtained.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc), was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc (Synthesis of Cyclized Poly 20mer)
  • Ser(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc, was obtained.
  • the target compound Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu), was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc (Synthesis of Cyclized Poly 21mer)
  • Tyr(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc, was obtained.
  • Step (2) Activation of Asp(OtBu)-Fmoc(D)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc (Synthesis of Cyclized Poly 22mer)
  • Asp(OtBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc (Synthesis of Cyclized Poly 23mer)
  • Ser(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc, was obtained.
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • Thr(tBu)-Fmoc In a vessel, 29.6 g of Thr(tBu)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Thr(tBu)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)Fmoc (Synthesis of Cyclized Poly 24mer)
  • Thr(tBu)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc (Synthesis of Cyclized Poly 25mer)
  • Phe-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc, was obtained.
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • Thr(tBu)-Fmoc In a vessel, 29.6 g of Thr(tBu)-Fmoc, 16.7 g of 1-hydroxy-1 H -benzotriazole hydrate, and 480 mL of dimethylformamide were added, and the mixture was completely dissolved by stirring. 15.5 mL of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution and stirred at room temperature for 30 minutes. Through the above process, the target compound, Thr(tBu)-Fmoc, was activated.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc (Synthesis of Cyclized Poly 26mer)
  • Phe-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc (Synthesis of Cyclized 27mer)
  • Gly-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc, was obtained.
  • Step (2) Activation of Gln(Trt)-Fmoc(Q)
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc (Synthesis of Cyclized 28mer)
  • Gln(Trt)-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc, was obtained.
  • Step (2) Activation of Aib-Fmoc
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc (Synthesis of Cyclized 29mer)
  • Aib-Fmoc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt) prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc (Synthesis of Cyclized 30mer)
  • His(Trt)-Boc activated in step (2) was added to the Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered.
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc, was obtained.
  • Example 5 Preparation of NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo[Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH 2 • TFA (Cleavage of Protecting Groups and Resins)
  • the temperature of the reaction solution of Vessel 1 was cooled to 5°C, and 2.0 L of methyl tertiary butyl ether, which is the cooled reactant in Vessel 3, was added thereto and stirred for 10 minutes.
  • the reaction solution was filtered and washed twice with 400 mL of the cooled methyl tertiary butyl ether.
  • the filtered crystals were dried under nitrogen atmosphere for 10 minutes.
  • the thus-dried crystals were added to Vessel 1, and 400 mL of distilled water was added thereto, and the mixture was stirred at room temperature for 10 minutes.
  • the reactant was filtered and washed with 600 mL of distilled water to obtain the target compound, NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo[Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH 2 • TFA.
  • Rink amide MBHA resin 80.0 g (0.31 mmol/g) of Rink amide MBHA resin and 480 mL of dimethylformamide were added to a vessel, stirred for 15 minutes, and then filtered to remove dimethylformamide. This process was performed twice. Through the above process, Rink amide MBHA resin was prepared.
  • Cys(Trt)-Fmoc activated in step (2) was added to the Fmoc-deprotected Rink amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and then filtered. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. 480 mL of methanol was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. 480 mL of dimethylformamide was added to the filtered resin, stirred for 2 minutes, and then filtered. This process was performed three times. Through the above process, the target compound, resin-Cys(Trt)-Fmoc, was obtained.
  • the target compound, Fmoc-deprotected resin-Cys(Trt), was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 1mer) prepared in Synthesis 1).
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • the target compound, Thr(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.7 g of Thr(tBu)-Fmoc.
  • the target compound, resin-Cys(Trt)-Thr(tBu)-Fmoc(C), was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu), was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 2mer) prepared in Synthesis 2).
  • Step (2) Activation of Asn(Trt)-Fmoc (N)
  • the target compound, Asn(Trt)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 29.6 g of Asn(Trt)-Fmoc.
  • the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Met-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 18.4 g of Met-Fmoc.
  • the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 4mer) prepared in Synthesis 4).
  • the target compound, Leu-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 17.5 g of Leu-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc (Synthesis of Poly 5mer)
  • the target compound, resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 5mer) prepared in Synthesis 5).
  • Trp(Boc)-Fmoc The target compound, Trp(Boc)-Fmoc, was activated in the same manner as in step (2) of Synthesis 1) with 26.1 g of Trp(Boc)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc (Synthesis of Poly 6mer)
  • the target compound, Gln(Trt)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 30.3 g of Gln(Trt)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc (Synthesis of Poly 7mer)
  • Step (2) Activation of Val-Fmoc(V)
  • the target compound, Val-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 16.8 g of Val-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc (Synthesis of Poly 8mer)
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 8mer) prepared in Synthesis 8).
  • Step (2) Activation of Phe-Fmoc(F)
  • the target compound, Phe-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.2 g of Phe-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Fmoc (Synthesis of Poly 9mer)
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 9mer) prepared in Synthesis 9).
  • Step (2) Activation of Glu(OtBu)-Fmoc(E)
  • the target compound, Glu(OtBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 21.1 g of Glu(OtBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Fmoc (Synthesis of Poly 10mer)
  • Step (2) Activation of Lys(Alloc)-Fmoc(K)
  • the target compound, Lys(Alloc)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 22.4 g of Lys(Alloc)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc (Synthesis of Poly 11mer)
  • the target compound, Ala-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 15.4 g of Ala-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Fmoc (Synthesis of Poly 12mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 12mer) prepared in Synthesis 12).
  • Step (2) Activation of Arg(Pbf)-Fmoc (R)
  • the target compound, Arg(Pbf)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 32.2 g of Arg(Pbf)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Fmoc (Synthesis of Poly 13mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Lys(Boc)-Fmoc(K)
  • the target compound, Lys(Boc)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 23.2 g of Lys(Boc)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Fmoc (Synthesis of Poly 14mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Glu(OAII)-Fmoc(E)
  • the target compound, Glu(OAII)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 20.3 g of Glu(OAII)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Fmoc (Synthesis of Poly 15mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Asp(OtBu)-Fmoc(D)
  • the target compound, Asp(OtBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 20.4 g of Asp(OtBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Fmoc (Synthesis of Poly 16mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Leu-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 17.5 g of Leu-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Fmoc (Synthesis of Poly 17mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Fmoc-deprotected resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu was obtained in the same manner as in step (1) of Synthesis 1) with the polypeptide (synthesis of poly 17mer) prepared in Synthesis 17).
  • the target compound, Tyr(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 22.8 g of Tyr(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc (Synthesis of Poly 18mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Lys(Boc)-Fmoc(K)
  • the target compound, Lys(Boc)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 23.2 g of Lys(Boc)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc (Synthesis of Poly 19mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Ser(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.0 g of Ser(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc (Synthesis of Poly 20mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Tyr(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 22.8 g of Tyr(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc (Synthesis of Poly 21mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Asp(OtBu)-Fmoc(D)
  • the target compound, Asp(OtBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 20.4 g of Asp(OtBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc (Synthesis of Poly 22mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Ser(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.0 g of Ser(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc (Synthesis of Poly 23mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • the target compound, Thr(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.7 g of Thr(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)Fmoc (Synthesis of Poly 24mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Phe-Fmoc(F)
  • the target compound, Phe-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.2 g of Phe-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc (Synthesis of Poly 25mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Thr(tBu)-Fmoc(T)
  • the target compound, Thr(tBu)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 19.7 g of Thr(tBu)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc (Synthesis of Poly 26mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, Gly-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 14.7 g of Gly-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc (Synthesis of Poly 27mer)
  • the target compound Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Gln(Trt)-Fmoc(Q)
  • the target compound, Gln(Trt)-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 30.3 g of Gln(Trt)-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc (Synthesis of Poly 28mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • Step (2) Activation of Aib-Fmoc
  • the target compound, Aib-Fmoc was activated in the same manner as in step (2) of Synthesis 1) with 16.1 g of Aib-Fmoc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc (Synthesis of Poly 29mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound, His(Trt)-Boc was activated in the same manner as in step (2) of Synthesis 1) with 30.7 g of His(Trt)-Boc.
  • Step (3) Preparation of Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAII)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc (Synthesis of Poly 30mer)
  • the target compound resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc, was obtained in the same manner as in step (3) of Synthesis 1).
  • the target compound NH 2 -Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo[Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-H • TFA was obtained in the same manner as in Example 4. [Table 1] Chromatogram Area (%) Example Comparative Example Specific gravity of maximum single related substance (%) 14.4 28.6 Crude purity (%) 61.6 18.0 Final yield after purification (%) 15 2
  • the box indicates the peak of the main product
  • the arrow indicates the peak position of the maximum single related substance
  • the blue circle indicates the chromatogram area where the peak corresponding to the maximum single related substance of Comparative Example appears.
  • RRT relative retention time
  • the Example according to the present invention showed a much improved result (improved by about 7.5 times) compared to the Comparative Example of the prior art in which cyclization was performed after linear synthesis.
  • the specific gravity of related substances in the process was reduced by half relative to the maximum single related substance (see ratio and crude purity of maximum single related substances of Table 1).
  • the maximum single related substance of the Comparative Example had an RRT of 1.12, but the related substance of the Example had an RRT of 0.86, which was further away from the main product peak (marked in the box of FIG. 1 ) in the chromatogram, and changed to a material which could be easily separated.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
EP20756094.7A 2019-02-15 2020-02-14 Neuartiges zwischenprodukt für biologisch aktives polypeptid und verfahren zur herstellung davon Pending EP3939990A4 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20190017766 2019-02-15
PCT/KR2020/002084 WO2020167010A1 (ko) 2019-02-15 2020-02-14 생리활성 폴리펩타이드에 사용되는 신규한 중간체 및 이의 제조방법

Publications (2)

Publication Number Publication Date
EP3939990A1 true EP3939990A1 (de) 2022-01-19
EP3939990A4 EP3939990A4 (de) 2023-05-24

Family

ID=72043917

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20756094.7A Pending EP3939990A4 (de) 2019-02-15 2020-02-14 Neuartiges zwischenprodukt für biologisch aktives polypeptid und verfahren zur herstellung davon

Country Status (4)

Country Link
US (1) US20220242913A1 (de)
EP (1) EP3939990A4 (de)
KR (1) KR20210118857A (de)
WO (1) WO2020167010A1 (de)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7928058B2 (en) * 2006-02-22 2011-04-19 Merck Sharp & Dohme Corp. Pharmaceutical composition comprising oxyntomodulin derivatives and a method for reducing body weight using the composition
RU2625793C2 (ru) * 2011-12-23 2017-07-19 Ипсен Мэньюфэкчеринг Айэлэнд Лимитед Способ синтеза терапевтических пептидов
KR101968344B1 (ko) * 2012-07-25 2019-04-12 한미약품 주식회사 옥신토모듈린 유도체를 포함하는 고지혈증 치료용 조성물
KR101993393B1 (ko) * 2012-11-06 2019-10-01 한미약품 주식회사 옥신토모듈린 유도체를 포함하는 당뇨병 또는 비만성 당뇨병 치료용 조성물
CA2890324C (en) * 2012-11-06 2021-02-23 Hanmi Pharm. Co., Ltd. Liquid formulation of protein conjugate comprising the oxyntomodulin and an immunoglobulin fragment
CN116063453A (zh) 2015-12-31 2023-05-05 韩美药品株式会社 胰高血糖素/glp-1/gip受体三重激动剂

Also Published As

Publication number Publication date
EP3939990A4 (de) 2023-05-24
KR20210118857A (ko) 2021-10-01
US20220242913A1 (en) 2022-08-04
WO2020167010A1 (ko) 2020-08-20

Similar Documents

Publication Publication Date Title
KR101087859B1 (ko) 인슐린친화성 펩타이드 합성법
CA2001303C (en) Endothelin dna and use thereof
TW202043260A (zh) 製備gip/glp1雙重促效劑之方法
EP2632935B1 (de) Verfahren zur herstellung von degarelix und seinen zwischenprodukten
WO2005090388A1 (en) Alpha helical mimics, their uses and methods for their production
JP2010514728A (ja) 環状ペプチドの合成方法
CN104428310A (zh) 细胞穿透肽和鉴定细胞穿透肽的方法
CZ296014B6 (cs) Způsob vazby zbytku aminokyseliny na peptidový řetězec
EP3414257B1 (de) Verfahren zur herstellung von liraglutide mithilfe eines bal-linker
CN113748125A (zh) 胰高血糖素样肽-1(glp-1)受体激动剂及其类似物的制备方法
CN109180803B (zh) 生长抑素及其制备方法和药物组合物
FI74711C (fi) Foerfarande foer framstaellning av terapeutiskt anvaendbara cykliska hexapeptidfoereningar.
JP2022527041A (ja) プレカナチドを製造する改善された方法
EP3939990A1 (de) Neuartiges zwischenprodukt für biologisch aktives polypeptid und verfahren zur herstellung davon
EP3914605B1 (de) Peptidfällungsverfahren
Dalcol et al. Convergent solid phase peptide synthesis: an efficient approach to the synthesis of highly repetitive protein domains
Taniguchi et al. O‐Acyl isopeptide method’for peptide synthesis: Solvent effects in the synthesis of Aβ1–42 isopeptide using ‘O‐acyl isodipeptide unit
KR20190134657A (ko) 펩타이드의 제조방법
LUU et al. Automated multiple peptide synthesis: Improvements in obtaining quality peptides
WO2021007703A1 (en) A method for preparing liraglutide via a solid phase peptide synthesis
US20230044268A1 (en) An improved process for preparation of liraglutide
CN113372429A (zh) 人血清淀粉样蛋白Aβ1-42的制备方法
CN113173987B (zh) 一种合成利司那肽的方法
Cavallaro et al. Solid‐phase synthesis of a dendritic peptide related to a retinoblastoma protein fragment utilizing a combined boc‐and fmoc‐chemistry approach
WO2023196765A1 (en) Process for preparing a glp-1/glucagon dual agonist

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20210915

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
RAV Requested validation state of the european patent: fee paid

Extension state: KH

Effective date: 20210915

A4 Supplementary search report drawn up and despatched

Effective date: 20230425

RIC1 Information provided on ipc code assigned before grant

Ipc: C07K 1/10 20060101ALI20230419BHEP

Ipc: C07K 1/06 20060101ALI20230419BHEP

Ipc: C07K 7/08 20060101ALI20230419BHEP

Ipc: C07K 7/64 20060101AFI20230419BHEP

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230523